Abstract
Human Factor IX is a highly post-translationally modified protein that is an important clotting factor in the blood coagulation cascade. Functional deficiencies in Factor IX result in the bleeding disorder haemophilia B, which is treated with plasma-derived or recombinant Factor IX concentrates. Here, we investigated the post-translational modifications of human serum-derived Factor IX and report previously undescribed O-linked monosaccharide compositions at serine 141 and a novel site of glycosylation. At serine 141 we observed two monosaccharide compositions, with HexNAc1Hex1NeuAc2 dominant and a low level of HexNAc1Hex1NeuAc1. This O-linked site lies N-terminal to the first cleavage site for the activation peptide, an important region of the protein that is removed to activate Factor IX. The novel site is an N-linked site in the serine protease domain with low occupancy in a non-canonical consensus motif at asparagine 258, observed with a HexNAc4Hex5NeuAc2 monosaccharide composition attached. This is the first reported instance of a site of modification in the serine protease domain. The description of these glycosylation events provides a basis for future functional studies and contributes to structural characterisation of native Factor IX for the production of effective therapeutic biosimilars and biobetters.
Highlights
Human Factor IX is a single chain zymogen that circulates in the plasma and plays an integral role in the coagulation pathway that stems bleeding at sites of vascular injury [1]
Asn residues within N-linked consensus sites can be assigned as unmodified or modified after peptides are detected by mass spectrometry (MS)
Almost complete sequence coverage was obtained for the serine protease domain which is not predicted or reported to contain any post-translational modifications (PTMs) (Fig. 1b)
Summary
Human Factor IX is a single chain zymogen that circulates in the plasma and plays an integral role in the coagulation pathway that stems bleeding at sites of vascular injury [1]. Factor IX is extensively modified in the endoplasmic reticulum (ER) and Golgi apparatus including: removal of the pre-pro leader sequence, γ-carboxylation, β-hydroxylation, phosphorylation, sulfation and N- and O-linked glycosylation [2,3]. Current treatments for haemophilia B include infusions of serum-derived or recombinant proteins, the latter of which is the preferred treatment in high-income countries [6,7,8,9]. Glycans are naturally heterogeneous due to non-template driven biosynthetic processing and branching of the glycosidic linkages [27] Both native and recombinant glycoproteins can be observed as different glycoforms with varying structures and degrees of site occupancy [28]. Unlike the N-linked glycosylation sequon, there is no amino acid consensus sequence that predicts likely sites for the O-glycosylation of Ser and Thr residues. We observed one novel Nglycosylation site, which, to our knowledge, has not been previously described
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